Von Willebrand factor (vWF) is a polydispersed multimeric plasma glycoprotein, which participates in the initial platelet adhesion and transport of coagulation FVIII. The size distribution of vFW multimers has a critical effect on its function, since the larger the vWF multimer, the more effective it is in promoting platelet adhesion. However, if ultra large multimers are present, spontaneous platelet aggregation and adhesion can occur and produce a thrombophilic state.
Recent studies have shown that ADAMTS13 (A Disintegrin-like And Metalloprotease with ThromboSpondin type 1 motif), a proteolytic enzyme synthesized primarily in the liver, is responsible for proteolysis of vWF within its A2 domain. A deficiency of ADAMTS13 will result in the presence of uncleaved ultra large vWF multimers, a circumstance not commonly observed in normal plasma [Levy, G. G. et al. Nature 413:488-494 (2001)]. In addition, these studies suggest that levels of activity and inhibition of this metalloprotease may be useful in the diagnosis and treatment of patients with thrombotic thrombocytopenia purpura (TTP) [Mannucci P. et al. 98:2730-35 (2001)]. There are several different manifestations of ADAMTS13 abnormalities. In congenital TTP, ADAMTS13 is found to be quantitatively deficient. With acquired TTP, an IgG autoantibody is formed that inhibits ADAMTS13. In other forms of acquired TTP/HUS (hemolytic uremic syndrome), ADAMTS13 may be present in variable concentrations. For example, deficiencies of ADAMTS 13 can result from E. Coli infections in children, veno-occlusive disease in bone marrow transplant, and a wide variety of drug toxicities.
The laboratory diagnosis and subtyping of vWF has been challenging since vWF does not appear on routine diagnostic blood tests. Physicians therefore must order specialized diagnostic blood tests to determine the specific vWF variant in order to establish the best and safest treatment for each patient.
Several types of assays currently exist for diagnosing vWF abnormalities. In the use of such assays, the vWF antigen must be determined for proper diagnosis. These assays variously include: radioimmunoassay (RIA) involving competitive binding of radiolabeled antigen and unlabeled antigen to a high-affinity antibody; enzyme immunoassay (EIA) and enzyme-linked immunosorbent assay (ELISA) [see U.S. Pat. No. 5,202,264] employing color reaction products of enzyme substrate interaction to measure antigen-antibody reaction; and latex immunoassay (LIA) utilizing antibodies bound at their Fv region to latex particles and presenting a Fab region for interaction with antigens present in blood samples [see U.S. Pat. No. 5,585,278].
The foregoing immunoassays are simple and widely used, but suffer several disadvantages. The immunoassays require labeled antibodies, which can be quite expensive and entail intrinsic hazards when radioactive labels are used. In addition, the occurrence of non-specific binding of proteins to antigens, the formation of antibody complexes, and the presence of various types of commonly used solid supports, can each increase background noise and reduce sensitivity, with the result that false-positive determinations are made [Harlow, E. Antibodies: A Laboratory Manual, Cold Springs Harbor Laboratory 1988].
Traditionally, abnormalities in vWF associated with disease states are further confirmed and classified into subtypes by multimeric structure and size distribution analysis of vWF using Western Blot electrophoresis. In principle, as many as 40 different molecular weight polymers are possible based on a dimer Mr of 500,000 and an upper limit molecular weight of 20 million. Current assessment of vWF multimer size distribution using Western Blot electrophoresis shows 10 to 15 distinct major electrophoretic mobilities. This classical and current method is used to detect epitopes of electrophoretically separated subspecies of antigens resulting in a spectral distribution of multimers by electrophoretic mobility. Electrophoresis of known molecular weight standards allows for the determination of the molecular weight of each antigenic band to which antibodies may be produced [Colman R W. Hemostasis and Thrombosis, Basic Principles and Clinical Practice, 4th edition; pp 825-837]. The disadvantages of this method can include: its substantial cost; its typical requirement of 3-5 days for completion of analysis, the need for a reporter group to label anti-vWF antibodies with radioactivity, fluorescence, or chemiluminescence for identification of multimers; and its reduced resolution of high and ultra-large molecular weight multimers.
Further, vWF related diseases involve the persistent diagnostic problem of non-specific bleeding symptoms. Current diagnostic testing for vWF is of limited value when low values for vWF:RCO (ristocetin co-factor) and vWF:Ag are present, due to the broad range of normal values of plasma vWF concentration. Compounding the difficulty is the fact that bleed times have an even wider normal variation than vWF levels. Therefore, the previously known tests do not provide the high level of precision that is essential to the accurate and reliable diagnosis of vWF related diseases.
Currently, there is a strong need for diagnostic tests that would be cost-effective, efficient, and provide additional molecular information benefiting the physician, laboratory and patient.